The androgen receptor (“AR”) is a ligand-activated transcriptional regulatory protein that mediates induction of male sexual development and function through its activity with endogenous androgens. Androgens are generally known as the male sex hormones. The androgenic hormones are steroids, which are produced in the body by the testes and the cortex of the adrenal gland or synthesized in the laboratory. Androgenic steroids play an important role in many physiologic processes, including the development and maintenance of male sexual characteristics such as muscle and bone mass, prostate growth, spermatogenesis, and the male hair pattern (Matsumoto, Endocrinol. Met. Clin. N. Am. (1994) 23:857–75). The endogenous steroidal androgens include testosterone and dihydrotestosterone (“DHT”). Testosterone is the principal steroid secreted by the testes and is the primary circulating androgen found in the plasma of males. Testosterone is converted to DHT by the enzyme 5 alpha-reductase in many peripheral tissues. DHT is thus thought to serve as the intracellular mediator for most androgen actions (Zhou, et al., Molec. Endocrinol. (1995), 9:208–18). Other steroidal androgens include esters of testosterone, such as the cypionate, propionate, phenylpropionate, cyclopentylpropionate, isocarporate, enanthate, and decanoate esters, and other synthetic androgens such as 7-Methyl-Nortestosterone (“MENT”) and its acetate ester (Sundaram et al., “7 Alpha-Methyl-Nortestosterone(MENT): The Optimal Androgen For Male Contraception,” Ann. Med., (1993) 25: 199–205 (“Sundaram”)). Because the AR is involved in male sexual development and function, the AR is a likely target for effecting male contraception or other forms of hormone replacement therapy.
Steroidal ligands, which bind the AR and act as androgens (e.g. testosterone enanthate) or as antiandrogens (e.g. cyproterone acetate), have been known for many years and are used clinically (Wu 1988). Although nonsteroidal antiandrogens are in clinical use for hormone-dependent prostate cancer, nonsteroidal androgens have not been reported.
Prostate cancer is the most frequently diagnosed non-skin cancer in American men, accounting for approximately 27% of all cancer cases (Boring, Cancer Statistics (1993), 43:7–26). Treatment for prostate cancer depends on the stage at which the cancer is found, and on the age and health status of the patient. As with other malignancies, accurate staging of prostate cancer is absolutely critical in selecting the most appropriate form of therapy. Clinically localized disease is potentially curable with standard surgery and/or radiation therapy. However, no curative therapies exist for advanced disease. Existing diagnostic tests such as magnetic resonance imaging (“MRI”), computed tomographic scans (“CT”), and ultrasound (“US”) lack both the specificity and sensitivity to substitute for exploratory surgery in staging malignant disease. Because of inadequate diagnostic studies, patients with what is presumed to be surgically curable prostate cancer must subrnit to surgical staging to determine the presence or absence of lymph node metastases. Almost half of the men initially diagnosed with local disease are found to have tumors which have advanced to the periprostatic area or beyond at the time of surgery (Carter et al., In: “A Multidisciplinary Analysis of Controversies in the Management of Prostate Cancer,” Coffey et al., eds., pp. 1–7, Plenum, New York). Thus, nearly one-third of all men diagnosed with prostate cancer (i.e., about 80,000 men per year) undergo surgery from which they are unlikely to benefit. Thus, non-invasive, more selective, and more accurate imaging tools for prostate cancer are needed.
Knowledge of the presence, location, and extent of disease aids in selecting which patients are likely to benefit from radical surgery, radiotherapy, or androgen ablation. Single photon emission computed tomography (“SPECT”) is a form of chemical imaging in which emissions from radiopharmaceuticals, labeled with gamma-emitting radionuclides (e.g. 99mTc, 67Ga, 111In, or 123I) are used to create cross-sectional CT images of radioactivity distribution in vivo. Imaging of this type is typically done with non-specific compounds (e.g. albumin or chelating agents like DTPA) complexed with the radionuclide. This, and similar methods, thus have the potential to improve the ability of CT and MRI to detect lymph nodes as well as bony and other visceral metastases. However, these methods are not selective. Radioimrnunoscintigraphy using monoclonal antibodies (“MoAbs”), which recognize prostate-specific proteins, including PSA and prostatic acid phosphatase (“PAP”), was developed as a means to specifically image the tumor, as opposed to the underlying host tissues. This approach has met with some success for these purposes (Dillman et al., “Radioimrnunodetection of Cancer With The Use of Indium-111-Labeled Monoclonal Antibodies,” Natl. Cancer Inst. Monogr., (1987), 3, 33; Vihko et al., “Immunoscintigraphic Evaluation of Lymph Node Involvement in Prostate Carcinoma,” Prostate (1987), 11, 51). Prostascint is an IgG1 murine monoclonal antibody conjugated to the 111In chelator GYK-DTPA to form the immunoconjugate 111In capromab pendetide. This antibody conjugate is reported to have a high degree of binding to all prostate cancers and mild binding to benign prostatic hypertrophic and normal prostate tissue. Preliminary data showed that this MoAb was able to detect disease foci of 5 mm or greater with a negative predictive value of 83% and positive predictive value of 50%, suggesting that radioimaging is a promising technique for prognostication of prostate cancer (Babaian et al., “Radioimmunoscintigraphy of Pelvic Lymph Nodes With 111-Indium-Labeled Monoclonal Antibody CYT-356,” J. Urol., (1994), 152, 1952). However, a major concern is that patients may develop human antimurine antibody (“HAMA”) responses as a result of the murine origin of the antibody, resulting in adverse reactions and precluding the use of subsequent antibody imaging (Babaian et al., “Radioimmunological Imaging of Metastatic Prostate Cancer With 111-Indium-Labeled Monoclonal Antibody PAY276,” J. Urol., (1987), 137, 439).
Chemical imaging represents a viable alternative to immunological methods. In this instance, the increase in imaging specificity is gained as the result of the preferential distribution of a radiochemical into an anatomical region of interest. Wolf recently defined pharmacokinetic imaging as the measurement of the rate of change of a radiochemical in an anatomic space, or chemical imaging with the added dimension of time (Wolf, “Imaging Can Be Much More Than Pretty Pictures,” Pharmaceut. Res., (1995), 12:1821–22). This technique can be powerful, because it allows one to obtain noninvasive measurements of the pharmacokinetics and pharmacodynamics of a radiolabeled compound at the target tissue site (Presant et al., “Association of Intratumoral Pharmacokinetics of Fluorouracil With Clinical Response,” The Lancet, (1994) 343:1184–87; Dowell et al., “Pharmacokinetic Parameters of Cisplatin Can Be Estimated in Human Tumors by Noninvasive Measurements of the Biodistribution and Targeting of 195mPt cisplatin,” Proc. Am. Assoc. Cancer Res., (1995); 36:360). Receptor-mediated chemical imaging has been used for the imaging of other endocrine tumors. 111In pentetreotide (Octreoscan®) is used clinically for the imaging of somatostatin receptors present in neuroendocrine tumors. It has also been shown that 16.alpha.-[18F] fluoroestradiol and 21-[18F]fluoro-16.alpha.-ethyl-19-norprogesterone can be used with positron emission tomography (“PET”) to provide clear images of both estrogen and progesterone receptor-positive breast tumors (Mintun et al., “Positron Tomographic Imaging of Estrogen Receptors in Human Breast Tumors,” Radiology, (1988), 169:45; McGuire et al., “Positron Tomographic Assessment of 16.alpha.-[18F]-Fluoroestradiol Uptake in Metastatic Breast Carcinoma,” J. Nucl. Med., (1991), 32:1526; Pomper et al., “21-[18F]fluoro-16.alpha.-ethyl-19-norprogesterone: Synthesis and Target Tissue Selective Uptake of a Progestin Receptor Based Radiotracer for Positron Emission Tomography,” J. Med. Chem., (1988), 31:1360; Dehdashti et al., “Assessment of 21-[18F] fluoro-16.alpha.-ethyl-19-norprogesterone as a Positron-Emitting Radiopharmaceutical for Detection of Progestin Receptors in Human Breast Carcinomas,” J. Nucl. Med., (1991), 32:1532). A variety of steroidal androgens incorporating photon-emitting and positron-emitting radionuclides have been synthesized and evaluated for their potential in imaging AR-positive tumors of the prostate (Carlson et al., “A Comparative Study of the Selectivity and Efficiency of Target Tissue Uptake of Five Tritium-labeled Androgens in the Rat,” J. Steroid Biochem., (1990), 36:549 (“Carlson”); Brandes et al., “Fluorinated Androgens and Progestins: Molecular Probes for Androgen and Progesterone Receptors with Potential Use in Positron Emission Tomography,” Molec. Pharmacol., (1987), 32:391 (“Brandes”); Liu et al., “20-[18F] fluoro-mibolerone, A Positron-Emitting Radiotracer For Androgen Receptors: Synthesis and Tissue Distribution Studies,” J. Nucl. Med., (991), 32:81 (“Liu 1991”); Choe et al., “Synthesis of 11.beta.-[18F] fluoro-5.alpha.-dihydrotestosterone and 11.beta.[18F] fluoro-19-nor-5.alpha.-dihydrotestosterone: Preparation Via Halofluorination-Reduction, Receptor Binding, and Tissue Distribution,” J. Med. Chem., (1995), 38:816 (“Choe”); Liu et al., “Synthesis of High Affinity Fluorine-Substituted Ligands for the Androgen Receptor. Potential Agents for Imaging Prostatic Cancer By Positron Emission Tomography,” J. Med. Chem., (1992), 35:2113 (“Liu 1992”); Hoyte et al., “7.alpha.-methyl-17.alpha.-(E-2′-[125I]iodovinyl)-19-nortestosterone: A New Radioligand for the Detection of Androgen Receptor,” Steroids (1993) 58:13 (“Hoyte”); Ali et al., “Synthesis of 17.alpha., 20E/Z)iodovinyl testosterone and 19-nortestosterone Derivatives as Potential Radioligands for Androgen and Progesterone Receptors,” J. Steroid Biochem. Mol. Biol., (1994), 49:15 (“Ali”)). However, the majority of these compounds were not useful for AR-mediated imaging due to rapid metabolic cleavage of the radiolabel, low AR binding affinity, or inadequate specific activity. Carlson and Katzenellenbogen examined the target tissue selectivity of tritiated testosterone, dihydrotestosterone, 19-nortestosterone, mibolerone (MEB), and methyltrienolone (R1881) in rats, concluding that compounds with AR binding affinities comparable to or greater than that of testosterone would be required to provide adequate target tissue uptake and target-to-nontarget contrast for successful in vivo imaging of androgen target tissues (Carlson). MIB and R 1881 demonstrated encouraging selectivity and target tissue uptake in animal models, most likely due to their slower in vivo metabolic clearance as compared to the other androgens (Carlson; Brandes; Liu 1992; Choe; Liu 1991). Bonasera (“Preclinical Evaluation of fluorine-18-labeled Androgen Receptor Ligands in Baboons,” J. Nucl. Med., (1996), 37:1009–15 (1996)) studied 18F-labeled steroids using PET in baboons and is now studying 16beta-[18F]fluoro-5.alpha.-dihydrotestosterone in men with metastatic prostate cancer.
Based on previous reports, the most important properties of radiolabeled androgens with respect to AR imaging appear to be: (i) the selectivity and affinity for AR binding; and (ii) the rate of in vivo metabolism (Carlson; Brandes; Liu 1992; Choe; Liu 1991; Hoyte; Ali). Androgenic steroids, like other steroids, are known to bind with other steroid receptors (Carlson; Dunn et al., “Transport of Steroid Hormones: Binding of 21 Endogenous Steroids to Both Testosterone-binding Globulin and Corticosteroid-binding Globulin in Human Plasma,” J. Clin. Endrocrinol., (1981) 53:58 (“Dunn”)). Binding of steroidal AR-imaging agents to progesterone and/or glucocorticoid receptors in the body contributes to their poor target site specificity for imaging. Radioactivity levels that remain in the blood or in non-target tissues are affected by the extent to which the agent binds to high affinity, non-target proteins (Carlson; Choe; Ali). Further, the natural androgens (i.e., testosterone and dihydrotestosterone) are extensively bound to sex hormone-binding globulin (“SHBG”), a high affinity, low capacity binding plasma protein (Dunn). Not surprisingly, many of the synthetic androgens also bind to SHBG with high affinity (Carlson; Brandes; Liu 1991; Liu 1992; Choe; Hoyte; Ali). These high affinity SHBG sites compete with the AR for specific binding of the radiolabeled ligand, and have precluded the use of a number of steroidal AR ligands for imaging. The metabolic fate and pharmacokinetics of AR-imaging agents are also important factors determining their usefulness for in vivo imaging (Carlson). Studies with estrogen and progestin imaging agents showed that in vivo target-to-nontarget uptake ratios correlated with the ratio of specific to non-specific binding in vitro (vanBrocklin et al., “16.beta.-([18F]fluoro)estrogens: Systematic Investigation of a New Series of Fluorine-18-Labeled Estrogens as Potential Imaging Agents for Estrogen-Receptor-Positive Breast Tumors,” J. Med. Chem., (1993), 35:1619). However, it was recently found that this relationship did not hold for a series of androgen analogs (Choe). In the series of compounds therein, the compound with highest relative binding affinity (“RBA”) and lowest non-specific binding had a-poor target-to-nontarget tissue uptake ratio, while the compound with the lowest RBA demonstrated the highest target-to-nontarget tissue ratio in vivo. As a whole, these data strongly suggest that in vivo metabolism is a major factor in determining the distribution profile in vivo (Carlson; Brandes; Choe; Ali).
In addition to their use in medical imaging, radiolabeled Androgen Receptor ligands (AR ligands) labeled with radionuclides (e.g. 90Y, 177Lu, 149Pm, 153Sm, 166Ho, 131I, 32P, 211At 47Sc, 109Pd, 105Rh, 186/188Re, 99Tc and 67Cu) may be used in therapy. In this case, the radionuclide (i.e., radioisotope) serves as the radiation source. The choice of the appropriate isotope for radiotherapy requires consideration of a variety of factors, such as tumor uptake and retention, blood clearance, rate of radiation delivery, half-life and specific activity of the radionuclide, and the economics of producing the radiopharmaceutical containing the radionuclide. The therapeutic AR ligand is designed to deliver the requisite amount of radiation dose to the tumor cells and to achieve a cytotoxic effect while avoiding side effects to normal tissue.
There is a need in the art to develop new non-steroidal compounds which bind the androgen receptor with high affinity and which are useful in imaging prostate cancer and in treating or preventing prostate cancer.